New triazole-based coordination complexes as antitumor agents against triple negative breast cancer MDA-MB-468 cell line

The present work describes the synthesis of a new triazole based ligand 3-(3,5-dimethyl-1H-pyrazol-1-yl)-1-methyl-1H-1,2,4-triazole (LM) and demonstration of its coordination diversity giving rise to a family of seven new coordination complexes, namely: [Ni(LM)3](ClO4)2·C2H6OS (5), [Co2(LM)6](ClO4)4·(C2H5)O (6), [Cd(LM)2Cl2] (7), [Cu(LM)2NO3]NO3 (8), [Fe(LM)3](BF4)2 (9), [Zn(LM)3](BF4)2 (10) and [Zn(LM)2NO3]NO3 (11), whose crystal structure was determined by single-crystal X-ray diffraction. Cytotoxic activity was evaluated against the MDA-MB-468 cancer cell line, which serves as a model for triple-negative breast cancer, and compared to the precursor molecule (L), as well as their coordination complexes (H3O){[NiL3](ClO4)3} (1), [CoL3](ClO4)2·2H2O (2), [CdL2Cl2] (3) and [CuL3](NO3)2 (4), for which the crystal structure was earlier determined. Notably, cadmium complexes 3 and 7 exhibit remarkable cytotoxicity and demonstrated a high selectivity index towards cancer cells when compared to peripheral blood mononuclear cells. Such activity highlights their potential function as anticancer agents.


Introduction
Breast cancer represents a signicant worldwide health concern, affecting millions of women each year. 1,2Triplenegative breast cancer (TNBC) stands out as a particularly aggressive and invasive type of cancer, and is considered to be the most difficult one to be treated effectively. 3It is characterized by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression. 4According to Cancer Treatment Centers of America (CTCA), TNBC accounts for approximately 10-15% of all breast cancer cases, presenting unique clinical and biological features that contribute to its aggressive nature and very limited treatment options.][7] Notwithstanding the development of intense therapeutic techniques, chemotherapy medications remain an important tool in the ght against breast cancer. 8,9They are also regarded as the cornerstones of cancer therapeutic research. 10Current TNBC treatment techniques are still inadequate, and polychemotherapy is the usual treatment for early TNBC in order to assess tumor sensitivity. 52][13] As a result, any advancement of new or additional chemotherapeutic solutions involves a highly challenging underlying chemistry.][16] Coordination complexes can exhibit enhanced anticancer activity compared to individual ligands or metal ions alone.8][19][20] This enhanced stability allows coordination complexes to effectively interact with biological targets, such as cancer cells, and exert their anticancer effects.Furthermore, the introduction of metal ions into coordination complexes can introduce additional mechanisms of action that are not present in the ligands alone.Metal ions can interact with cellular components, such as DNA or proteins, leading to specic biological effects.These interactions can disrupt cellular processes, induce apoptosis, inhibit cell proliferation, or interfere with signaling pathways crucial for cancer cell survival and growth.
2][23] A number of new chemicals containing a 1,2,4-triazole scaffold were developed and tested for anticancer activity against a panel of cancer cell lines. 24Furthermore, some highly promising outcomes were achieved in the case of 1,2,4-triazole-3-thiol derivatives against human melanoma IGR39, human triplenegative breast cancer (MDA-MB-231) and pancreatic carcinoma (Panc-1) cell lines.Their potential selectivity towards the same cells was also investigated. 25Interestingly, a large number of triazole based molecules demonstrated their efficiency against TNBC. 26Among those, Azeliragon triazole analogues have recently been identied for their excellent activities. 27][30][31] Herein, we report the syntheses and characterization a new triazole based ligand 3-(3,5-dimethyl-1H-pyrazol-1-yl)-1-methyl-1H-1,2,4-triazole (LM) (Scheme 1), that was characterized by different spectroscopic techniques, including 1 H NMR, 13  The structural features of these coordination complexes were investigated using single-crystal X-ray diffraction.Moreover, these complexes were evaluated against the MDA-MB-468 cancer cell line, which was used as a model for the triplenegative breast cancer.The precursor L and its coordination complexes ( 4) that we previously reported for their antibacterial and antifungal properties, 32 were also investigated and discussed for comparison.

Materiel and instrumentation
All solvents and chemicals, obtained from usual commercial sources, were of analytical grade and used without further purication. 1H and 13 C NMR spectra were obtained using a Bruker AC 300 spectrometer.High resolution mass spectrometry HRMS data were obtained with a Q Exactive Thermo-sher Scientic ion trap spectrometer by using ESI ionization.FT-IR spectra were recorded on KBr pellets using a Perki-nElmer 1310 spectrometer.UV-visible spectra were recorded using a Shimadzu 3600 plus spectrometer equipped with Harrick praying mantis modulus which allows direct analysis of powders in reectance mode.Elemental analysis was performed with a vario EL microanalyzer (C, H, N and O).Magnetic susceptibilities were measured on a Quantum design MPMS-5s SQUID magnetometer.Magnetic data were corrected for the sample holder and diamagnetic contributions.A polycrystalline sample of 9 was quickly loaded into a gelatine capsule and immediately inserted within the SQUID cavity to avoid any air oxidation.Mössbauer spectra were recorded in transmission geometry with a constant acceleration mode conventional spectrometer equipped with a 50 mCi 57 Co(Rh) source and a Reuter Stokes proportional counter.The powdered sample was sealed in plastic sample holder and a spectrum was recorded at 298 K.The spectrum was tted using Recoil 1.05 Mössbauer Analysis soware. 33Isomer shi values are given with respect to a-Fe at room temperature.

X-ray crystallography
Suitable single crystals were selected and mounted onto a rubber loop using Fomblin oil.Crystal data for 5, 6, 8, 9 and 10 have been collected on a MAR345 Image plate detector using a monochromated (montel optics) microfocus Mo Ka radiation (l = 0.71073 Å) source (Incoatec ImS).Data integration and reduction was performed using the CrysAlisPRO crystallographic soware package and the implemented absorption correction was used. 34rystal data for 7 have been collected on a Rigaku R-AXIS RAPID II diffractometer using graphite monochromated Mo Ka radiation (l = 0.71075 Å) and the u-f scan technique.All structures were solved employing dual space direct methods (SHELXT), 35 and rened by SHELXL2018/3. 36Nonhydrogen atoms were rened anisotropically with hydrogen placed in calculated positions and rened in riding mode.
Single-crystal X-ray diffraction data of 11 were recorded on a Bruker Apex CCD diffractometer (l (Mo Ka) = 0.71073 Å) at 150 K equipped with a graphite monochromator.Structure solution and renement were carried out with SHELXS-97, 37 and SHELXL-97, 38 using the WinGX soware package. 39Data collection and reduction were performed using the Apex2 soware package.Corrections for incident and diffracted beam absorption effects were applied using empirical absorption corrections. 40All atoms and most of carbon atoms were rened anisotropically.Solvent molecule sites were found and included in the renement of the structures.
Table S1 † provides the crystallographic data, data collection and renement details for the compounds.Table S2 (ESI †) contains a summary of selected bond lengths [Å] and angles [°].
2.3.8 [Zn(M) 2 NO 3 ]NO 3 (11).LM (35.4 mg, 0.2 mmol, 2 equiv.)was dissolved in methanol (3 mL).Zn(NO 3 ) 2 $4H 2 O (26.1 mg, 0.1 mmol, 1 equiv.)was also dissolved in methanol (3 mL) and added to the LM solution, and stirred for 10 min at room temperature.Colourless single crystals were obtained aer 10 days via vapour diffusion of diethyl ether (10 mL) at room temperature.Yield 32%.FT   4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay as described in our previous study. 41Briey, cells were seeded in 96-well microtiter plates at a density of 10 4 cells per well.Aer overnight incubation, cells were exposed to different concentrations (0-400 mM) of each compound in 100 mL of culture medium.The plates were then incubated.Negative control (DMSO) and positive control (paclitaxel) were included, ensuring the nal concentration of DMSO remained below 0.2%.Aer 48 h, 20 mL of MTT solution (5 mg mL −1 ) were added to each well, followed by a 4 h incubation.Subsequently, 150 mL of culture medium were replaced with 150 mL of isopropanol-HCl to dissolve the formazan crystals.All incubations were carried out in a humidied atmosphere at 37 °C and 5% CO 2 .The optical density was measured at l = 540 nm, and cell viability was determined by calculating the percentage of absorbance of treated cells relative to untreated cells.
2.4.2Study of cytotoxic activity on non-cancer cells.Blood samples from healthy volunteer donors were used aer approval by the Biomedical Research Ethics Committee.Peripheral blood mononuclear cells (PBMCs) were isolated using a density gradient method following the manufacturer's instructions (Capricorn Scientic).The PBMCs were then seeded into 96-well microtiter plates at a density of 2 × 10 4 cells per well.The cytotoxic effect was evaluated on PBMCs under the same conditions and concentrations as previously described for tumor cells using the MTT assay.
2.4.3Statistical analysis.Data were rst exported as an Excel le for initial analysis.Subsequently, viability values obtained were transferred to GraphPad Prism 8 for further analysis.A one-way ANOVA with Tukey's multiple comparisons test was conducted as a post hoc test.The data is presented as means ± SD from three independent experiments.Statistical signicance was determined at a p-value of less than 0.05 in all cases.

Synthesis of LM and its coordination complexes (5-11)
Considering our previously reported bioactive coordination complexes (1-4) using ligand L, 32 we have decided to enhance the potential of this ligand by a straightforward alkylation using iodomethane.The synthesis procedure we documented is centred on utilizing t-BuOK and THF instead of other options for the base and solvent.This choice was made because it enabled the extraction of exceptionally pure LM without the need for additional purication, resulting in a higher yield.
Our aim was to explore how the choice of metal and counter anions impacts the formation and structural characteristics of our coordination complexes, while also evaluating their potential as anticancer agents.To achieve this, we employed various ligand/metal ratios, tested diverse metal salts with varying counter anions, and employed different crystallization methods/solvents.Among the compounds we examined, 5-11 emerged as the most promising and yielded high-quality single crystals suitable for X-ray analysis.

FT-IR and UV-visible spectroscopies
Fig. 1 illustrates the FT-IR comparison plot of the ligand LM, and its relative coordination complexes.LM exhibits characteristic peaks located at 3131 and 3009 cm −1 both of which represent the N-H stretching vibration of triazole and pyrazole, respectively.Furthermore, due to the C-H aromatic vibration, a weak peak can be observed at 2965 cm −1 .Additionally, the peaks centred at 1557 and 1443 cm −1 are assigned to the -N]N and C]C aromatic stretching vibrations.It is undoubtable that most of the representative ligand-based vibration peaks were shied upon coordination with the transition metals.The biggest shi was mostly observed in the case of -N]N stretching vibrations, since all of the metals were coordinated in this segment of the ligands.Besides, the spectra of 5 and 6 with perchlorate counter anions revealed a new strong peak at 1095 and 1088 cm −1 , respectively. 42Likewise, new peaks identied in the FT-IR spectra of compound 8 and 11 around 1350 cm −1 can be assigned to the NO 3 − counter anion. 43g.S1 † shows the diffuse reectance spectroscopic comparison of LM and complexes 5-11.Bands in the UV region (∼200-350 nm) can be assigned to the intra-ligand transitions such as p-p* and n-p*.Moreover, complex 5, exhibits a weak band at l = 570 nm.This latter one corresponds to a d-d transition as earlier observed at l = 589 nm for the nickel complex [Ni(dpbmp) 2 ](ClO 4 ) 2 with dpbmp = diethyl-1,1 ′ -(pyridine-2,6diyl)bis(5-methyl-1H-pyrazole-3-carboxylate). 44Complex 6 also displayed one band at l ∼ 490 nm, associated to a d-d transition in an octahedral surrounding. 45On the other hand, copper complex 8 also exhibited a large band around 730 nm, that can be ascribed to a d-d transition, aligning with the distorted octahedral structure usually observed in Cu(II) complexes. 46The cadmium 7 and the zinc 10, 11 complexes do not adsorb in the visible range. 47,48

Single crystals X-ray analysis
Compound 5 crystallizes in the triclinic system, space group space P 1 (#2).The crystal structure shows a nickel mononuclear complex coordinated with three LM ligand through their nitrogen atoms (Fig. 2).The coordination geometry is distorted octahedral, with bond angles [:N-Ni-N = 77.18(9)-100.49(9)°] containing six nitrogen atoms of three bidentate LM ligands.The bond lengths between the Ni(II) and the nitrogen from the pyrazole rings, range from 2.136(2) to 2.142(2) Å.The bond lengths between the Ni(II) and the nitrogen from the triazole ring was found to be shorter and ranges between 2.072(2) and 2.080(2) Å.Two distorted perchlorate counter anions with a DMSO solvent molecule are also present in the asymmetric unit.also slightly shorter when compared to N p -Co pyrazole bonds (2.170(2)-2.173(2)Å).Two perchlorate counter anions identied in the unit cell, in addition to a distorted diethyl ether can be spotted in the crystal lattice.Compound 9 adopts a crystalline structure within the triclinic system, in P 1 space group.In a similar fashion to 5 and 6, the structure reveals a distorted octahedral complex with angles falling within the range [:N-Fe-N = 74.03(8)-102.94(8)°],while two distorted non coordinated tetrauoroborate anions are present in the crystal lattice.The bond length between Fe II and nitrogen atoms is relatively long ranging from 2.152(2) to 2.216(2) Å, which fall in the range of expected values for a high-spin (HS) state for Fe II ions. 49It is almost equal to the Fe-N bond lengths found for the polymeric 2D material, [Fe(btre) 2 (NCS) 2 ] (btre = 1,2-bis(1,2,4-triazol-4-yl) ethane), which is known to not be switchable neither by temperature, pressure or red light irradiation. 50Such long distance observed in 9 thus hardly precluded a HS to low-spin transition to occur.A magnetic measurement performed over the range (300-5 K) conrmed a full HS state (Fig. S2 † Complex 7 is a mononuclear cadmium complex which crystallizes in the monoclinic system, P 2 1 /c space group.The coordination geometry around Cd(II) is a slightly distorted octahedral arrangement, with bond angles of [:N-Cd-N = 89.24(6)-90.76(8)°].Additionally, the distances between Cd and the nitrogen atoms in the two triazole rings are equal (2.411(3) Å) each, and the same applies to the N-Cd bond lengths with the two pyrazole rings, with both 2.414(2) Å.In a comparable manner to [CdL 2 Cl 2 ] (3), 7 is coordinated to two bidentate LM ligands in the equatorial positions and two chloride anions in the axial positions with the same Cd-Cl distance of 2.543(9) Å and the Cl-Cd-Cl bond angle is perfectly linear (180.0°).
Complex 8, crystallizes in the monoclinic system, C 2/c space group.The structure shows a mononuclear copper complex coordinated with two bidentate LM ligands.The Cu(II) coordination sphere adopts a distorted octahedral arrangement, characterized by bond angles measuring [:N-Cu-N = 79.22(9)-104.36(13)°],involving four nitrogen's from the two bidentate LM ligands, as well as two oxygen from the coordinated nitrate counter anion in apical position.The bond distances between the metal and oxygen, triazolic and pyrazolic nitrogen atoms are 2.018(5), 2.112(2) and 2.042(2) Å, respectively, which contrasts to the trend observed with the rest of complexes where a longer N t -Cu bond length was observed with the triazolic nitrogen when compared to the N p -Cu bond length with the pyrazolic one.Compound 11, also crystallizes in the C 2/c space group, with a mononuclear Zn(II) complex coordinated in a octahedral fashion to four nitrogen atoms from two bidentate chelating LM

Cytotoxicity against MDA-MB-468
The viability of MDA-MB-486 tumour cells was assessed using the MTT assay aer a 48 h treatment with varying concentrations of L, LM and their metal complexes 1-11.Both ligands L  and LM did not exhibit any effect, in contrast to our metal complexes, in particular 3, 4 and 7 (Fig. 3).
Such enhanced cytotoxicity suggests that coordination of the ligands with specic metal ions resulted in a synergistic effect that amplied their anticancer properties, facilitating their interactions with cellular components and promoting cellular uptake.Interestingly, complexes 3 and 7 demonstrated remarkable cytotoxicity against tumor cells, with complex 4 showing also superior characteristics.At concentrations below 60 mM, the cadmium complexes completely destroyed the cells, indicating a potent anticancer effect.
In order to validate these ndings, it is essential to assess the cytotoxic activity of the metal complexes on non-cancer cells, which could help determine their safety and selectivity index (Fig. 4).Table 1 highlights the inhibitory concentration 50 (IC 50 ) in mM and selectivity index of L, LM ligands and their metal complexes against MDA-MB-486 cancer cell line and normal PBMCs cells.Whereas 4 displayed a selectivity index of 0.97, indicating a lack of reliability as a TNBC antitumor drug, 3 and 7, presented selectivity index values of 1.34 and 1.30, respectively.These results suggest that 3 and 7 hold greater potential as TNBC anticancer agents, with outstanding IC 50 values of 15.61 and 19.90, respectively, against the MDA-MB-486 cancer cell line.Remarkably, 3 and 7 are both made of only two ligands, leaving two positions occupied by independent chlorine atoms, which are made available to better interact with cellular components, leading to such benecial properties.

Conclusion
In conclusion, the reaction of LM, with assorted metal salts led to the formation of a family of seven novel coordination complexes with diverse coordination environments.This further conrms the versatility of 1,2,4-triazole derivatives in coordination chemistry.Most importantly, the evaluation of MDA-MB-486 tumor cell viability following treatment with varying concentrations of ligands L, LM, and their metal complexes revealed intriguing ndings.The coordination complexes exhibited signicantly improved performance compared to their respective ligands, indicating that the ligand-metal coordination has a profound effect on the cytotoxic activity of the resulting complexes.Although cadmium alone has shown to promote the proliferation of TNBC in numerous reported studies, 51,52 the newly emerged cadmium complexes 3 and 7 not only demonstrated effective inhibition against MDA-MB-468 cell lines but also showcased a selectivity index exceeding 1.This conclusively demonstrates a contrary effect to proliferation.Moreover, it is important to note that exposure to cadmium can result in diverse impacts on human health, including kidney damage, cardiovascular effects, and bone damage.Consequently, it will be necessary to conduct thorough investigations into the safety and potential drawbacks of these novel compounds.Despite the signicant gap in the literature regarding the development of efficient coordination materials for triple negative breast cancer, which is known to be a challenging form of tumor cells, this pioneering study paves the way for exciting opportunities and avenues to explore.Further investigations into the underlying mechanisms of action and in vivo studies are warranted to fully understand the therapeutic potential and safety prole of these coordination complexes for cancer treatment, as well as the syntheses of new complexes.

2. 4 .
Anticancer activities 2.4.1 Study of cytotoxic activity on cancer cells.The in vitro antitumor activity of L and LM, along with their metal complexes 1-4 and 5-11 respectively, was assessed against the MDA-MB-468 breast cancer cell line, following the [3-(

Fig. 3
Fig. 3 Viability of MDA-MB-486 tumor cells following 48 h treatment with different concentrations of L, LM and their metal complexes 1-11, evaluated by MTT assay.Results are means ± SD from three independent experiments.Paclitaxel was used as reference compound.

Fig. 4
Fig. 4 Viability of PBMC cells following 48 h treatment with different concentrations of L, LM and their metal complexes, evaluated by MTT assay.Results are means ± SD from three independent experiments.Paclitaxel was used as a reference material.

Table 1
Inhibitory concentration 50 (IC 50 ) in mM and selectivity index of L, LM ligands and their metal complexes against MDA-MB-486 cancer cell line and normal PBMCs cells a Each value represents the average of three separate experiments.Distinct letters denote signicant differences between the treatments (p < 0.05).b IC 50 (PBMCs)/IC 50 (MDA-MB-468). a